Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

Increased Ca2+ content of the sarcoplasmic reticulum provides arrhythmogenic trigger source in swimming-induced rat athlete's heart model.

Sudden cardiac death among top athletes is very rare, however, it is 2-4 times more frequent than in the age-matched control population. In the present study, the electrophysiological consequences of long-term exercise training were investigated on Ca2+ homeostasis and ventricular repolarization, together with the underlying alterations of ion channel expression, in a rat athlete's heart model. 12-week swimming exercise-trained and control Wistar rats were used. Electrophysiological data were obtained by using ECG, patch clamp and fluorescent optical measurements. Protein and mRNA levels were determined by the Western immunoblot and qRT-PCR techniques. Animals in the trained group exhibited significantly lower resting heart rate, higher incidence of extrasystoles and spontaneous Ca2+ release events. The Ca2+ content of the sarcoplasmic reticulum (SR) and the Ca2+ transient amplitude were significantly larger in the trained group. Intensive physical training is associated with elevated SR Ca2+ content, which could be an important part of physiological cardiac adaptation mechanism to training. However, it may also sensitize the heart for the development of spontaneous Ca2+ release and extrasystoles. Training-associated remodeling may promote elevated incidence of life threatening arrhythmias in top athletes.

Inotropic effect of NCX inhibition depends on the relative activity of the reverse NCX assessed by a novel inhibitor ORM-10962 on canine ventricular myocytes.

Na+/Ca2+ exchanger (NCX) is the main Ca2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca2+ ([Ca2+]i). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1µM ORM-10962 reduced the Ca2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca2+]i. L-type Ca2+ current (ICa) was not affected by 1µM ORM-10962 in the absence of SR Ca2+ release, while ICa was suppressed by ORM-10962 during normal Ca2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca2+]i, suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca2+i]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca2+ handling and/or the preserved inducibility of forward NCX by high [Ca2+]i. This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure.

The investigation of the cellular electrophysiological and antiarrhythmic effects of a novel selective sodium-calcium exchanger inhibitor, GYKB-6635, in canine and guinea-pig hearts.

The sodium-calcium exchanger (NCX) is considered as the major transmembrane transport mechanism that controls Ca2+ homeostasis. Its contribution to the cardiac repolarization has not yet been directly studied due to lack of specific inhibitors, so that an urgent need for more selective compounds. In this study, the electrophysiological effects of GYKB-6635, a novel NCX inhibitor, on the NCX, L-type calcium, and main repolarizing potassium currents as well as action potential (AP) parameters were investigated. Ion currents and AP recordings were investigated by applying the whole-cell patch clamp and standard microelectrode techniques in canine heart at 37 °C. Effects of GYKB-6635 were studied in ouabain-induced arrhythmias in isolated guinea-pig hearts. At a concentration of 1 µmol/L, GYKB significantly reduced both the inward and outward NCX currents (57% and 58%, respectively). Even at a high concentration (10 µmol/L), GYKB-6635 did not change the ICaL, the maximum rate of depolarization (dV/dtmax), the main repolarizing K+ currents, and the main AP parameters. GYKB-6635 pre-treatment significantly delayed the time to the development of ventricular fibrillation (by about 18%). It is concluded that GYKB-6635 is a potent and highly selective inhibitor of the cardiac NCX and, in addition, it is suggested to also contribute to the prevention of DAD-based arrhythmias.

Antiarrhythmic potential of drugs targeting the cardiac ryanodine receptor Ca2+ release channel: case study of dantrolene.

Driven by the limitations of the traditional antiarrhythmic pharmacology, current antiarrhythmic research is trying to identify new avenues for the development of specific and safe antiarrhythmic drugs. One of the most promising approaches in this field is the amelioration of the abnormal events in cellular Ca(2+) handling originating from the dysfunction of ryanodine receptor Ca(2+) release complex (RyR), which is an inevitable key factor in the pathology of myocardial dysfunction, remodeling and arrhythmogenesis. Accordingly, both in experimental and clinical situations, inhibition of abnormal activity of RyR, regardless of being the primary cause or a consequence during the pathogenesis appears to exert beneficial effect on disease outcome, including a marked antiarrhythmic defense. Considerable amount of our knowledge in this field originates from studies using dantrolene, a human drug with RyR stabilizing effect. Our review summarizes the cardiovascular pharmacology of dantrolene and the results of its use in experimental models of cardiac diseases, which emphasize a promising perspective for the possible antiarrhythmic application of RyR inhibition in the future.